Ciliary neurotrophic factor (CNTF) is a protein that is required for the survival of embryonic chick ciliary ganglion neurons in vitro (Manthorpe et al., 1980, J. Neurochem. 34:69-75). The ciliary ganglion is anatomically located within the orbital cavity, lying between the lateral rectus and the sheath of the optic nerve; it receives parasympathetic nerve fibers from the oculomotor nerve which innervates the ciliary muscle and sphincter pupillae.
Over the past decade, a number of biological effects have been ascribed to CNTF in addition to its ability to support the survival of ciliary ganglion neurons. CNTF is believed to induce the differentiation of bipotential glial progenitor cells in the perinatal rat optic nerve and brain (Hughes et al., 1988, Nature 335:70-73). Furthermore, it has been observed to promote the survival of embryonic chick dorsal root ganglion sensory neurons (Skaper and Varon, 1986, Brain Res. 389:39-46). In addition, CNTF supports the survival and differentiation of motor neurons and hippocampal neurons. (International Application No. PCT/US 90/05241)
Recently, CNTF has been cloned and synthesized in bacterial expression systems, as described in copending U.S. application No. 07/570,651, entitled "Ciliary Neurotrophic Factor," filed Aug. 20, 1990 by Sendtner et al. incorporated by reference in its entirety herein.
In addition to CNTF, the receptor for CNTF (originally termed "CNTFR") has been cloned, sequenced and expressed (see copending U.S. application No. 07/700,677, entitled "The Ciliary Neurotrophic Factor Receptor," filed May 15, 1991 and now abandoned by Davis, et al. and International Application No. PCT/US91/03896, filed Jun. 3, 1991 which are incorporated by reference in their entirety herein).
Unlike other known growth factor receptors which have an extracellular domain, a hydrophobic transmembrane domain and a cytoplasmic domain, the CNTF receptor does not appear to have a cytoplasmic domain. Furthermore, it is linked to the cell surface via a covalent linkage from the protein to an oligosaccharide which is in turn glycosidically linked to phosphatidylinositol (referred to as a "GPI-linkage"). GPI-linkages play a role in the attachment of proteins, such as alkaline phosphatase (APase) to membranes. The role of GPI linkages in the function of biological membrane components have been elucidated as a major means of anchoring proteins to biological membranes in the case of at least 30 distinct proteins. [(Slein, et al., J. Bacteriol. 80:77 (1960); Low, et al., Biochemistry 19:3913 (1980)].
Treatment of the cell-surface membrane of CNTF responsive cells with phosphatidylinositol-specific phospholipase C (PI-PLC) releases CNTFR from the cell membrane. Such release is prohibited, however, if CNTF is first bound to the receptor prior to exposure to the enzyme, indicating a possible interaction between CNTF, its receptor and a third, signal transducing component.
The discovery that CNTFR and CNTF may form a complex that interacts with a membrane bound, signal transducing component suggested therapeutic activity of a soluble CNTF/CNTFR receptor complex. In copending application U.S. Ser. No. 801,562 filed Dec. 2, 1991 by Yancopoulos, et al. entitled "Cell Free Ciliary Neurotrophic Factor/Receptor Complex", which is incorporated by reference in its entirety herein, CNTF and CNTFR are combined to form a stable, biologically active complex that can be used as a differentiation or proliferation factor in cell types that express signal transducing receptor components belonging to the CNTF/IL-6/LIF receptor family.
One such signal transducing component involved in high affinity binding of CNTF and the subsequent functional response of the cell has been identified as gp130, a .beta. component common to the IL-6, Oncostatin M, LIF family of receptors. A further .beta. component identified as being involved in binding and signal transduction in response to LIF (LIFR.beta.) appears to be the same or similar to a .beta. component necessary for response to CNTF. (As a consequence of the identification of .beta. components necessary for binding and signal transduction of CNTF, what was originally generally termed CNTFR is currently referred to as CNTFR.alpha.). Accordingly, as described in U.S. application Ser. No. 07/865,878 filed on Apr. 8, 1992 and now abandoned entitled "Cell-Free Ciliary Neurotrophic Factor/Receptor Complex", which is incorporated by reference in its entirety herein, signal transduction can be initiated by treatment of cells expressing both gp130 and LIFR.beta. with a soluble CNTF/CNTFR.alpha. complex. Alternatively, target cells not previously responsive to CNTF, but expressing LIFRI.beta. and gp130 (such as LIF-responsive cells) can be made responsive to CNTF by attaching the CNTFR.alpha. to the cells and subsequently treating with CNTF.
The molecular cloning of the coding region for human CNTFR.alpha. (hCTNFR.alpha.) enabled the generation of probes useful for detecting the presence of receptor message in a variety of tissues. The results of such studies indicated that CNTFR.alpha. mRNA was detectable in tissues of the central nervous system, sciatic nerve, adrenal tissue and in skeletal muscle. As described in copending application U.S. Ser. No. 07/700,677, now abandoned, entitled "CNTF Receptor" which is incorporated by reference in its entirety herein, a detailed analysis of the CNTF receptor expression in muscle indicated that the CNTF receptor is expressed in both myotube and myoblast muscle cell lines of either mouse or rat origin, as well as in both red slow-twitch soleus muscle and white fast-twitch extensor digitorum longus (EDL) muscle of the rat.
Further described in U.S. application No. 07/700,677, now abandoned, is the finding that CNTF receptor mRNA was increased in both soleus and EDL muscle in animals that were first denervated for 72 hours relative to their sham-operated contralateral controls.